Properties of an intergenic terminator and start site switch that regulate IMD2 transcription in yeast.

Abstract

The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of approximately 200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

Sequence of the IMD2 promoter region. Initiation sites in the presence of guanine are indicated by thin bent arrows. The initiation site when guanine is limiting is depicted by the thick bent arrow (44). Map positions at which poly(A) is added to IT-terminated, intergenic RNAs are circled. The TATA box (per Escobar-Henriques et al. [13]) and start codon are shown in large type. The IT is identified with a solid underline (single and double). The portion of the IT used to create a chimeric terminator is doubly underlined. The hexamer mutagenized to a BsiWI site (AGTATG → CGTACG, where the mutated nucleotides are underlined) is boxed. The consensus Nab3 binding site is in a shaded box.

Exosome sensitivity of the IT- and CYC-terminated transcripts. RNA was prepared for Northern analysis from yeast strains with the wild-type RRP6 gene or deleted for RRP6 and bearing a plasmid containing the GAL1 promoter and either the CYC1 or IMD2 intergenic terminator (IT) inserted downstream of the promoter (indicated schematically at the bottom of the figure). The strains were DY1038 (RRP6) (lanes 1 to 3), DY1403 (Δrrp6) (lanes 4 to 6), DY1532 (RRP6) (lanes 7 to 9), DY1534 (Δrrp6) (lanes 10 to 12), DY1035 (RRP6) (lanes 13 to 15), and DY1400 (Δrrp6) (lanes 16 to 18). Cells were induced with 2% galactose for 0, 30, or 90 min and probed with the GAL1 or SED1 (as a loading control) probe, as indicated.

Exosome sensitivity of terminated transcripts. RNA was prepared for Northern analysis from yeast strains with the wild-type RRP6 gene or deleted for RRP6 and bearing a plasmid containing the GAL1 promoter and either the CYC1 terminator, the IT, or a chimera between the CYC1 and IMD2-IT. The chimera contained 49 bp of the IT (double underlined in Fig. 1) placed 5′ to 102 bp of the CYC1 core terminator (there was a 4-bp overlap of common sequence between them). Terminators were inserted at proximal (XbaI in panel A) or distal (BsiWI in panel B) positions downstream of the promoter (indicated schematically at the bottom of the figure). In panel A, the strains were DY1550 (RRP6) (lanes 1 to 3), DY1551 (Δrrp6) (lanes 4 to 6), DY1532 (RRP6) (lanes 7 to 9), D1534 (Δrrp6) (lanes 10 to 12), DY1038 (RRP6) (lanes 13 to 15), and DY1403 (Δrrp6) (lanes 16 to 18). In panel B, the strains were DY1554 (RRP6) (lanes 1 to 3), DY1555 (Δrrp6) (lanes 4 to 6), DY1558(RRP6) (lanes 7 to 9), and DY1559 (Δrrp6) (lanes 10 to 12)]. Transcription was induced following galactose exposure for the indicated times. Filters were probed with the GAL1 or SED1 probe as indicated. Full-length (IT in the reverse orientation in the XbaI site) marker RNA from galactose-induced strain DY1552 was loaded in the lane marked M in panels A and C. In panel C, transcription through an IT with a mutated consensus Nab3 binding site (AGUU) or an unaltered site (UCUU) was analyzed by Northern blotting as described above for panels A and B.

SEN1 dependence of termination. RNA was prepared for Northern analysis from wild-type yeast and strains with the Sen1E1597K mutation. Each strain contained plasmids with the GAL1 promoter and either the CYC1 terminator or the IMD2 IT inserted downstream of the promoter. Terminators were inserted at promoter-proximal (XbaI) or promoter-distal (BstEII) positions. The strains used in panel A were DY682 (SEN1) (lanes 1 to 4), DY686 (sen1E1597K) (lanes 5 to 8), DY684 (SEN1) (lanes 9 to 12), and DY688 (sen1E1597K)(13 to 16). The strains used in panel B were DY2900 (SEN1) and DY2902 (sen1E1597K). The strains were grown at the indicated temperatures (25°C or 37°C) after 0 or 90 min of galactose induction, and the filters were probed with the GAL1 or SED1 probe as indicated. Trace levels of an unstable transcript are indicated by the unlabeled black arrow to the left or right of the gel.

Effect of spacing upon start site selection. (A) Start site mapping of IMD2 with increasing spacing between the “high-guanine” and “low-guanine”' (MPA) starts. An rrp6Δ strain was transformed with a family of plasmids containing increasing amounts of lambda DNA inserted into the engineered BsiWI site (boxed in Fig. 1) between the high-G and low-G start sites. Each derived strain was grown in the presence (+) of 0.5 mM guanine for 30 min or 15 μg/ml MPA for 2 h before RNA was prepared and subjected to 5′-end mapping by primer extension. The strains used were DY1701 (lanes 1 and 2), DY1702 (lanes 3 and 4), DY1703 (lanes 5 and 6), DY1704 (lanes 7 and 8), DY1706 (lanes 9 and 10), DY1707 (lanes 11 and 12), and DY1708 (lanes 13 and 14). Extension products representing the low-guanine/downstream and high-guanine/upstream starts are indicated. Phosphorimaging was used to quantify the extension product corresponding to the MPA-induced start and plotted as a function of the length of DNA inserted between the two starts. Prism 4.0 (GraphPad Software, Inc.) was used for curve fitting. (B) Northern blot of IMD2 transcripts with increasing spacing between the “high-guanine” and “low-guanine” starts. RNAs from strains described in panel A were analyzed by Northern blotting. Strains DY1700 (lanes 1 to 3), DY1701 (lanes 4 to 6), DY1702 (lanes 7 to 9), DY1703 (10 to 12), DY1704 (lanes 13 to 15), DY1706 (lanes 16 to 18), DY1707 (lanes 19 to 21), and DY1708 (lanes 22 to 24) were grown in synthetic complete medium lacking uracil (SC−ura) with 0.5 mM guanine and 15 μg/ml MPA for 2 h before RNA was prepared. Filters were probed with the “upstream” or “downstream” probes described in Materials and Methods. wt, wild type; Bsi, BsiWI. (C) Cultures from the strains in panel B were grown to an optical density at 600 nm of 0.01 and serially diluted in fourfold increments, and 5 μl of each dilution was spotted onto SC-ura and SC-ura containing 15 μg/ml MPA and grown at 30°C. WT, wild type.

Effect of Rpb9 subunit on initiation site selection. (A) Start site mapping. RNA transcribed from the native chromosomal loci was analyzed by Northern blotting from strains deleted for RRP6, RPB9, or both genes, as indicated in the figure. Strains DY1549 (lanes 1 to 3), YSC1021-551682 (lanes 4 to 6), and BY4741-4437 (lanes 7 to 9) were grown in the presence of 0.5 mM guanine for 30 min or 15 μg/ml MPA for 2 h before RNA was isolated for primer extension. Some cells received neither treatment (− lanes). The fast moving band in all lanes is a nonspecific species in the probe preparation that serves as an internal standard. (B) Northern analysis. RNAs from strains BY4741-4437 (lanes 1 to 3), YSC1021-551682 (lanes 4 to 6), and DY1549 (lanes 7 to 9) were prepared as described above for panel A and subjected to Northern blotting. The top panel of the Northern blot was probed with the “upstream” probe described in Materials and Methods, and the lower panel was probed with the SED1 probe as a loading control.